Scientific research has led to extensive developments in cell-based and tissue-based therapies. In recent years, with the advancement of regenerative medicine and tissue engineering, fundamental studies and treatments using living cells have been performed widely.
The field of regenerative medicine is focussed on regenerating damaged tissues and organs in the body by replacing damaged tissue and/or by stimulating the body's own repair mechanisms to heal previously irreparable tissues or organs. This often involves the use of cells or tissue, in cell or tissue therapy, either from the same person (autologous) or from another donor (allogeneic). There are many potential forms of cell therapy, including: the transplantation of stem cells or progenitor cells; the transplantation of mature, functional cells; and the application of modified human cells that are used to produce a needed substance (cell-based gene therapy).
Cell based therapy is targeted at many indications, both medical and non-medical, and in multiple organs and tissues using several modes of cell delivery. Accordingly, the specific mechanisms of action involved are wide ranging. However, there are two main principles by which cells facilitate their desired action:                i) Stem cell or progenitor cell engraftment, differentiation, and long term replacement of damaged tissue. In this paradigm multipotent or unipotent cells differentiate into a specific cell type in the laboratory or after reaching the target site, where they integrate to replace lost tissue, and thus facilitate regeneration and renewal.        ii) Cells that have the capacity to release cellular factors such as cytokines, chemokines, growth factors or proteins. The delivered cells (via local or systemic administration) remain viable for a relatively short period (days-weeks) and then die. This includes cells that naturally secrete the relevant factors, or which undergo epigenetic changes or genetic engineering that causes the cells to release large quantities of a specific molecule. Examples of this include cells that secrete factors which facilitate angiogenesis, anti-inflammation, and anti-apoptosis.        
Whilst it is widely recognized that such cell-based transplantations have application in the medical field to alleviate disease conditions and disorders, current research has led to the increased application of such techniques for non-medical purpose, including use in the cosmetic industry. Many recent cosmetic products and techniques are based on advanced scientific research that includes the use of biotechnology-derived ingredients, nutritional regimens, stem-cell-based products and therapies to regenerate ageing tissues, or use cell and tissue engineering for cosmetic purposes.
Through improved understanding of the structure of the skin and its underlying repair and maintenance processes, researchers are increasingly able to intervene to reduce the effects of premature ageing, improve healing processes or ‘enhance’ the appearance of skin. The cosmetics and pharmaceutical industries have also expended considerable effort to understand the ageing processes of the skin and to devise countermeasures.
In relation to point ii) above, a recent development is the use and transplantation of one particular skin cell type, the melanin producing cells called melanocytes, which are found in the bottom layer (the stratum basale) of the skin's epidermis. Melanin is the pigment primarily responsible for skin colour, produced through a process called melanogenesis. Numerous cosmetic conditions, or cosmetic effects as a consequence of an underlying disorder, can result in generalized or localized hyperpigmentation (increased skin colour) and hypopigmentation (reduced skin colour) of the skin. Commonly, in the case of localized hypopigmentation, this is attributed to partial or complete loss of melanin such as experienced in:                Pityriasis alba: a sequelae of eczema in which asymptomatic oval pink scaly patches resolve to leave pale macules for some months or longer. Reduced numbers of active melanocytes and a decrease in number and size of melanosomes are seen in affected skin for unknown reasons;        Treatment-induced hypopigmentation: Medications and treatments used to treat various skin conditions can result in lightening of the skin. These include dermabrasion, use of chemical peels, and local steroid injections;        Postinflammatory hypopigmentation (leukoderma): as a consequence of many inflammatory skin conditions, such as skin infection, blisters, burns, cryotherapy, dermal injury with scarring or eczema, loss of pigmentation may occur in the affected area;        Vitiligo: a condition characterised by chronic and progressive depigmentation of areas of the skin. Vitiligo occurs when melanocytes in the basal layer of the epidermis are defective or die. The cause of vitiligo is unknown, but research suggests that different factors might act independently or synergistically to determine the phenotype, including autoimmunity, genetic susceptibility, viral infection, environmental factors, or oxidative stress;        Piebaldism: a rare autosomal dominant disorder of melanocyte development and migration. Common characteristics include a congenital white forelock, scattered normal pigmented and hypopigmented macules and a triangular shaped depigmented patch on the forehead;        Alezzandrini syndrome: a very rare syndrome characterized by a unilateral degenerative retinitis, followed after several months by ipsilateral vitiligo on the face and ipsilateral poliosis; and        Melanoma-associated leukoderma: a cutaneous condition characterized by vitiligo-like depigmentation that can occur in patients with cutaneous or ocular melanoma.        
Whilst the loss of pigmentation observed in the likes of the above may be of a purely cosmetic concern and only secondary to the underlying pathology leading to same, often the cosmetic dysfunction has a significant negative impact on quality of life. The altered pigmentation is often immediately visible to others and individuals may suffer social and emotional consequences including low self-esteem, social anxiety, relationship problems and depression. Therefore, in addition to finding treatments for the underlying causes of the disease e.g. in the case of vitiligo, it is also paramount to the patient to consider the cosmetic aspects.
In addition to melanocytes, other skin cell types exist with similar potential utility.
Keratinocytes constitute around 90% of the cells of the epidermis, where at the skin surface they produce increasing amounts of keratin and synthesize and extrude lipids into the intracellular space. At the top of the epidermis the cells have differentiated to a cell type that are called corneocytes, which form the outermost skin layer and are constantly shed and replaced by new cells. This differentiation process is tightly controlled to maintain the integrity of the physical skin barrier.
It is known that epidermal melanocytes form a functional and structural unit with neighboring keratinocytes to stimulate melanocyte proliferation, with growth factors produced by adjacent keratinocytes regulating the proliferation and differentiation of melanocytes. Structural changes in keratinocytes may result in loss of melanocytes and evidence suggests that keratinocytes in depigmented epidermis are more vulnerable to apoptosis. Keratinocyte apoptosis will result in reduced expression of keratinocyte-derived factors in depigmented epidermis, resulting in melanocyte death. The relationship between keratinocytes and melanocytes will therefore clearly play a role in skin depigmentation, and the delivery of healthy keratinocytes to depigmented skin, possibly, although not necessarily, in combination with healthy melanocytes (or vice versa), represents a viable strategy for re-pigmentation.
Epidermal stem cells, such as those in the stratum basale or hair follicle, have attracted similar interest from the cosmetics industry and various companies are already exploring their potential for skin-care products, for example, skin regeneration and repair, or anti-ageing products that contain proteins derived from specialized stem-cell lines that affect specific receptors in both fibroblasts and keratinocytes that increase the production of collagen.
Dermal fibroblasts produce collagen (type I, type III and type VII), elastin, hyaluronic acid and matrix metalloproteinases and therefore are essential in forming elongated fibres and extracellular matrix. Dermal fibroblasts produce the structural components that unite separate cell layers and allow epithelial cells of the epidermis to join together to form upper skin barrier layers. The cells also allow skin to recover from injury. For this reason, transfer of dermal fibroblasts has been used cosmetically as anti-ageing and scar remodeling products. Autologous (self) fibroblasts are also being used to treat skin wrinkles, scars, folds and depressions and for lip augmentation. The advantage of using fibroblasts over direct collagen injection relates to the breaking down of collagen protein by endogenous enzymes.
Therefore, the potential for transplantation of cells is readily recognized in the field. A plethora of techniques have thus been developed for delivering cells. For example, to target depigmentation, the transfer and/or culture of target cells, such as keratinocytes, fibroblasts, or melanocytes, from pigmented (normal unaffected skin) to depigmented skin has been tested.
US20100310526 discloses a method for increasing or intensifying the pigmentation of skin, by the application of melanocyte precursor cells from hair root sheaths onto the depigmented area. It is disclosed that keratinocyte precursor cells are also applied to the depigmented area. The procedure requires the recipient site is dermabraded (surgical skin planing) before the application of a cell solution.
US20120064049 discloses a method for the regeneration of aged skin for cosmetic purposes and for the prevention of skin diseases, using stem cells from hair root sheaths and/or keratinocyte and melanocyte precursor cells. According to this method, before the application of the cell solution the epidermis at the recipient site needs to be physically or mechanically ablated, preferably by means of superficial dermabrasion, laser treatment (fraxel laser), or superficial needle puncture (dermaroller).
US20110150848 discloses a method for producing a transplantable cellular suspension of living tissue for grafting to a patient using the ReCell® Spray-On Skin™ system. The method requires harvesting donor tissue from the patient (4 cm2 biopsy, thickness 150-200 μm), performing in-theatre preparation of a spray-on suspension consisting of cells derived from the biopsy, and applying this suspension immediately over the recipient graft site, on a surface up to 80 times the size of the biopsy. The recipient site needs to be dermabraded or laser-treated before treatment. The ReCell suspension contains basal keratinocytes, melanocytes, fibroblasts and Langerhans cells. The metabolically responsive epithelial cells migrate across the wound surface, leading to regeneration of skin of normal colour and texture.
However it is apparent that the many existing methods for transferring tissue (skin grafting) or cells have associated inherent problems, including:                Koebner phenomenon: This is the development of disease at sites of trauma. For example if someone with psoriasis scratches themselves, they can develop the phenomenon along the skin that was scratched.        Scarring: The transfer of skin via grafts or punches leads to defects, trauma and scarring to skin at a second site.        Skin preparation: Cells can only be transplanted onto prepared skin, i.e. skin that has had its epidermis removed via invasive techniques such as dermabrasion.        Pain: These invasive techniques can be very painful and are not without risk.        Infection: Techniques such as dermabrasion cause major barrier defect and increase the risk of infection.        Skill: Methods are heavily dependent on the skill of a surgeon to carry out the procedure.        Recovery: current procedures require 1-2 weeks recovery.        
There is a real unmet need to find more efficient, pain free and cost-effective technologies to repair the skin and permit transfer of cells into same thus achieving a better cosmetic outcome (e.g. 80% or more re-pigmentation without scarring).
Heretofore it has not been shown that cells can be successfully delivered using microneedles. Unexpectedly, the inventors have determined that cells can be injected into surface layers of the skin using fine bore microneedles with no observed loss of viability. Moreover, using a similar such approach, it has been found that cells can be extracted from the skin and that these extracted cells are viable in culture.
The present invention therefore concerns a new minimally-invasive method for the repair of the skin comprising step a) the extraction and step b) the transfer and delivery of skin cells, such as melanocytes, fibroblasts or keratinocytes, using microneedles (MNs), including the targeting of a cellular suspension of freshly extracted, or cultured, cells to the skin. Notably, different layers of the skin can be targeted as the cells will reposition to the appropriate area.
Microneedles are micron-sized, needle-like projections often, but not always, organized in an array of a defined geometric pattern on a planar base plate. They are an established technology currently being exploited for the targeted intra-epidermal and intradermal delivery of drugs and vaccines. Due to their microscopic dimensions MNs do not penetrate skin deep enough to cause any significant pain, bleeding or scarring, as demonstrated through numerous clinical trials. Application of MNs to the skin surface results in penetration of the outer skin barrier, the stratum corneum (SC), and the creation of multiple transient micro-pathways that permit the delivery of materials without impinging significantly on nerves or blood vessels.
Most notably, the micron-scale dimensions of the microneedle shafts allow for simple and direct application into skin that does not require professional training.
A pilot study by a team at Cardiff University has shown the minimally invasive nature of microneedles, compared with conventional hypodermic injection, demonstrating that microneedles caused significantly less pain than normal needles. Further, advantageously, following withdrawal of the microneedle the induced disruptions in the skin surface rapidly reseal thus leaving minimal or no scarring and minimal barrier defect.
This disclosed method therefore paves the way for a new minimally-invasive and pain-free approach wherein cells can be extracted and delivered to the various layers of the skin using microneedles, with little or no recovery time required. There would be no need for highly trained surgeons and expensive equipment to perform the procedure, making the procedure more affordable. Additionally, the use of microneedles minimizes scarring with no perceived change in skin texture or pigment, and given the minimal invasiveness of the technology, the need for prior treatment of the targeted areas is circumvented.